Platinum-based therapies are used to treat a broad range of malignant diseases, but their efficacy is limited by development of resistance. Repair of DNA interstrand crosslinks (ICLs) induced by platinum-based compounds involves multiple DNA repair pathways including the Fanconi anemia (FA) and postreplication repair (PRR) pathways. Rad6 is essential for PRR as mutation of its ubiquitin conjugation site renders cells PRR-compromised. Recent studies suggest PRR pathway proteins Rad6, Rad18 and PCNA are pivotal in FancD2 activation, a critical event in FA pathway repair of ICLs. To determine the role of Rad6 in sensitivity and repair of platinum-induced damage, we used cancer cell models displaying intrinsic cisplatin (CDDP) resistance (metastatic MDA-MB-231 breast cancer cells, IC50 12.5 μM) and an isogenic model of acquired oxaliplatin (OxPt) resistance (parental HCT116, IC50 1.2 μM vs. resistant HCT116 (HCT116-OxR) colon carcinoma cells, IC50 12 μM). MTT assays showed that pretreatment with a Rad6 small molecule inhibitor increased sensitivities to CDDP (IC50 reduced from 12.5 to 2.8 μM) and OxPt (∼22% increased cytotoxicity at concentrations < 5 μM OxPt). Consistent with these data, immunoblot analysis indicated Rad6 inhibitor decreased CDDP induction of γH2AX, reflecting reduced repair and increased drug sensitivity. Although Rad6 protein levels were minimally affected by the inhibitor, levels of CDDP-induced monoubiquitinated-PCNA (indicating Rad6 TLS pathway activation) and CDDP-induced ubiquitinated-FancD2 (indicating FA pathway activation) were reduced. Immunofluorescence staining and subcellular fraction analysis indicated increased CDDP sensitivity in the presence of Rad6 inhibitor is associated with decreased CDDP-induced nuclear localization of γH2AX, FancD2, PCNA and Rad6. Orthotopic tumors produced by MDA-MB-231 cells pretreated with Rad6 inhibitor were significantly smaller compared to control, and tumor volumes were further reduced when combined with CDDP. A similar relationship between OxPt-induced Rad6 and FA pathway crosstalk was observed in HCT116-OxR cells. Rad18 protein was upregulated, and FancD2 and PCNA were constitutively monoubiquitinated in HCT116-OxR cells compared to parental HCT116 cells, in which FancD2 and PCNA ubiquitination was observed only after OxPt exposure. γH2AX was constitutively upregulated in HCT116-OxR cells. These data suggest OxPt tolerance is conferred by active damage signaling and repair. Rad6 inhibitor treatment decreased γH2AX, monoubiquitinated-PCNA and FancD2. These data imply a major role for the Rad6 PRR pathway in intrinsic and acquired platinum-based compound resistance and suggest inhibition of Rad6 ubiquitin conjugating activity may be exploited to alleviate resistance to platinum-based therapies. Supported by DOD W81XWH-09-1-0608, WSU Bridge Funding and KCI Strategic Research Initiative Grant.
Citation Format: Matthew A. Sanders, Malathy PV Shekhar. Exploring the role of the Rad6 pathway in tumor resistance to platinum-based compounds. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5505. doi:10.1158/1538-7445.AM2013-5505
Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.